Quantifying and modeling ligand-dependent control of RORγ dynamics via structural proteomics

核受体(NR)超家族的配体调节转录因子已被证明是一个丰富的来源。 开发多种人类疾病的治疗方法的目标。内源性小分子 这些变构蛋白的调节控制着哺乳动物生理学的大多数方面的中心过程。我们的 实验室专注于NRS的NR1F亚家族的合成配体开发和结构-功能分析 被称为维甲酸受体相关的孤儿受体，或RORS。这个亚家族包含三个基因 参与但不限于调节葡萄糖和脂肪代谢、骨骼生长和免疫。 功能。在这个建议中，我们试图扩大我们对NR1F3(RoRγ； 基因名称RORC)，在完整的全长受体的上下文中。RoRγ有两种亚型，即RoRγ1和 RoRγ2，它们只在N端序列上有所不同。RoRγ1广泛表达，在肝脏中发挥作用 在昼夜节律和糖脂代谢中起重要作用。RoRγ2或RoRγt的表达式为T 细胞特异性的，已被证明是启动分化的关键谱系定义转录因子 TH17细胞的程序使RoRγt成为TH17和Tc17分化的重要调节因子。重要的是，这些 已经显示出抗肿瘤效果的细胞和RoRγt控制程序的基因程序，增强免疫和 减少免疫抑制。我们已经报道了内源性的高亲和力的甾醇和含氧甾醇。 Ligands和其他人已经证实了这些发现，并提供了关键证据，表明它们确实是生理的 RoRγ配体。尽管在某些实验范例中，RoRγ可以在不添加 外源配体，提示该受体可能是结构性活性的。最近的证据清楚地表明 RoRγ的激活依赖于配体结合。 虽然对受体孤立结构域的广泛结构研究为高密度脂蛋白提供了重要的洞察力 对于激动剂和拮抗剂的亲和配体结合，目前还缺乏关于模数如何结合的信息 RoRγ的结构域在完整的全长受体的背景下共同作用。鉴于RoRγ作为 作为一个治疗靶点，令人惊讶的是，我们对小分子如何调节 它的活动。“关闭”RoRγ活动的机制看起来很简单；然而，我们有一个 对感受器是如何“启动”的理解不完全。我们假设配体依赖的结构 干扰影响受体的定位和PTM状态，影响其辅调节因子和DNA 相互作用对RoRγ转录组的调控。为了为检验这一假说提供基础，我们 建议开发和验证完整的RoR全长γ/DNA复合体的完整结构模型 扩大我们对RoRγ配体依赖调控的理解。阐明RoRγ激活机制 将有助于开发更好的工具来研究其药理学，并可能通过设计新的治疗策略 功能选择性配体。

The nuclear receptor (NR) superfamily of ligand regulated transcription factors has proven to be a rich source of targets for the development of therapeutics for a wide range of human diseases. Endogenous small molecule regulation of these allosteric proteins control processes central to most aspects of mammalian physiology. Our lab has focused on synthetic ligand development and structure-function analysis of the NR1F subfamily of NRs known as the retinoic acid receptor-related orphan receptors or the RORs. This subfamily contains three genes that are involved in but not limited to regulation of glucose and lipid metabolism, bone growth, and immune functions. In this proposal we seek to expand our understanding of ligand-dependent regulation of NR1F3 (RORγ; gene name RORC), in the context of the intact full-length receptor. There are two isoforms of RORγ, RORγ1 and RORγ2, that differ in only their N-terminal sequence. RORγ1 is broadly expressed, and in the liver it plays an important role in circadian rhythms and glucose and lipid metabolism. The expression of RORγ2 or RORγt, is T cell specific and has been shown to be the key lineage-defining transcription factor to initiate the differentiation program of TH17 cells making RORγt an essential regulator for TH17 and Tc17 differentiation. Importantly, these cells that have demonstrated anti-tumor efficacy and RORγt controls gene programs that enhance immunity and decrease immune suppression. We have reported sterols and oxygenated sterols as high affinity endogenous ligands and others have confirmed these findings and provided key evidence that they are indeed physiological RORγ ligands. Although in certain experimental paradigms RORγ can recruit coactivators without addition of exogenous ligand, suggesting the receptor may be constitutively active. Recent evidence clearly demonstrates that RORγ is dependent on ligand binding for activation. While extensive structural studies on isolated domains of the receptor have provided important insight into high affinity ligand binding for both agonists and antagonists, there is a lack of information on how the modular domains of RORγ act together in the context of the intact full-length receptor. Given the importance of RORγ as a therapeutic target, it is surprising that we have an incomplete understanding of how small molecules modulate its activity. The mechanism for “turning off” RORγ activity appears straightforward; however, we have an incomplete understanding on how the receptor is “turned on.” We hypothesize that ligand-dependent structural perturbations manipulate the localization and PTM status of the receptor influencing its coregulator and DNA interactions to modulate of the RORγ transcriptome. To provide the groundwork to test this hypothesis, we propose to develop and validate an integrated structural model of intact full-length RORγ/DNA complex to expand our understanding of ligand-dependent regulation of RORγ. Illuminating RORγ activation mechanisms will help develop better tools to study its pharmacology and may lead to new therapeutic strategies by designing functionally selective ligands.